Prepare 3 kinds of pad A, B and C provided by Hangzhou Xiaojiemei Sanitary Articles Co., Ltd., and 6 samples for each kind of pad. Cut the middle part of the pad as a rectangle with a length of 5cm and a width of 3cm (Figure 1)(the area is 15cm2).Immerse the cut rectangle into the beaker filled with water-glycerol mix until it fully absorbs water and sinks to the bottom of the beaker. Clamp a small corner of the rectangle with tweezers and lift it out of the water surface. When there is no water drop, clamp it out of the beaker and weigh the reduced weight of beaker with water-glycerol mix. Convert the reduced weight into the amount of mix absorbed by the rectangle.

Figure 1. The rectangle cutting, sample collection and process of Pad’s blood absorption experiment.

Cell lysis experiment

4 tubes of MDA-MB-435 cells were resuspended by 1mL PBS, mixed mildly and divided them into 8 tubes (500μL/tube). For experiment group, 250, 100 or 50μL ddH2O was added into the tube, then added PBS to 750μL. For negative control, 250μL PBS was added, for positive control, cells were lysed by ultrasonic.

CRISPR/Cas12a Biosensing System

RPA

Generally, 2μL forward and reversed primer (10μM) with 1μL template (1ng/μL) and 12.5μL Rehydration buffer were added into a tube with protein dry powder, add ddH2O to 47.5μL. Add 2.5μL of 280mM Magnesium Acetate (MgOAc) (supplied) and mix well to start reaction. RPA reaction was carried out in room temperature for 20min, unless otherwise described.

Primer selection experiment with PCR

Here are the primers we use:

Figure 2. The sequence of HPV16 L1 gene. Shows the primers.

Green Taq Mix (Vazyme) was used to amplify the target sequence with PCR. 2μL forward and reversed primer (10μM) with 1μL template (1ng/μL) were added into 25μL Green Taq Mix, and add ddH2O to 50μL. PCR annealing temperature for 1F-2R, 3R, 4R; 3F-2R, 3R, 4R and 4F-2R, 3R, 4R primer pairs are 57℃, for 2F-2R, 3R, 4R primer pairs are 60℃. After PCR, 10× DNA loading buffer (30% (v/v) glycerol, 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol) were added, the reaction mix was separated by 1.5% agarose gel pre-stained with SYBER Gold (Invitrogen).

Primer selection experiment with RPA

1F-4R, 3F-4R, 4F-2R, 3R, 4R primer pairs were used to amplify the target sequence. RPA protocol has been mentioned above. After the RPA finished, the reaction mix would either be stored at 4℃ for further experiments or be quenched with DNA loading buffer (30% (v/v) glycerol, 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol) containing 15 mM EDTA and separated by 1.5% agarose gel pre-stained with Gel red (TOYOBO).

70bp DNA reporter cleavage assay

Generally, 0.5μL crRNA (2μM) and 0.5μL Cas12a protein (1μM) were added directly into 7.6μL of RPA or PCR products with 0.4μL 10× Cas12a reaction buffer. Add 1μL reporter (70bp, 15μM), mix well to start cleavage. The reaction was incubated at 25℃ for 80min. Reactions were quenched with DNA loading buffer (30% (v/v) glycerol, 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol) containing 15 mM EDTA and separated by 1.5% agarose gel pre-stained with Gel red (TOYOBO).

Fluorophore quencher (FQ)-labelled reporter assay

The reaction mix was the same as DNA reporter cleavage assay. The only difference is that reporters are 5bp single strand DNA with 5’-FAM and 3’-TAMRA. For qualitative detection, an UV flashlight was used to check if FAM was emitted green florescence. For quantitative detection, a real-time fluorescence detection device (see hardware) was used, the reaction was carried out in a microﬂuidic chip or a specially made plastic plate with a small groove. Fluorescence was measured every minute.

Lateral flow readout of Cas12a activity using FAM-Biotin reporters

The reporter was a 5bp single strand DNA with 5’-Fitc and 3’-Biotin. After the cleavage reaction was carried out for 80 min, the mix was used to run on lateral flow strips.

Hybridization Chain Reaction Biosensing System

Preparation of the probe.

All probes (BGI Bio Tech.) were prepared in 1*NEBuffer 1 and heated at 90℃ for 10min, then they were allowed air cooling for 1 hour to form hairpin structure.

HPV genome digestion.

Add 2ng HPV L1 plasmid, 0.5μL of each endonuclease (FastDigest Restriction Enzyme, Thermo Fisher), 2U ExoIII, 2μL 10*NEBuffer 1, add ddH20 to the total volume of 20μL, and react in room temperature for 4h. Verify the result by 1% agarose gel pre-stained with Gel red (TOYOBO).

ExoIII assisted signal amplification.

Add 2μL P2 or P1-S2 (10μM), 2U ExoIII, 2μL 10*NEBuffer 1 and S1 of various concentrations, add ddH20 to the total volume of 20μL, and react in room temperature for 1h. Verify the result by 2% agarose gel pre-stained with Gel red (TOYOBO).

Hybridization chain reaction.

Add 2μL H1 (10μM), 2μL H2 (10μM), 1μL 10*NEBuffer 1 and S2 of various concentrations, add ddH20 to the total volume of 10μL, and react in room temperature for 4h. Verify the result by 5% or 8% native PAGE.

Lateral flow nucleic acid biosensor.

Add 100μL ddH20 to the hybridization chain reaction production, test the solution by test paper.

Combination of ExoIII assisted signal amplification and hybridization chain reaction.

Add 1μL P2 or P1-S2 (10μM), 2μL H1 (10μM), 2μL H2 (10μM), 2U ExoIII, 2μL 10*NEBuffer 1 and S1 of various concentrations, add ddH20 to the total volume of 20μL, and react in room temperature for 4h. Verify the result by 2% agarose gel pre-stained with Gel red (TOYOBO).

Combination of preprocessing of HPV genome, ExoIII assisted signal amplification and hybridization chain reaction.

Add 2ng HPV L1 plasmid, 0.5μL of each endonuclease (FastDigest Restriction Enzyme, Thermo Fisher), 2U ExoIII, 2μL 10*NEBuffer 1, 1μL P2 or P1-S2 (10μM), 2μL H1 (10μM), 2μL H2 (10μM), add ddH20 to the total volume of 20μL, and react in room temperature for 4h. Verify the result by 8% native PAGE.

Combination of preprocessing of HPV genome, ExoIII assisted signal amplification, hybridization chain reaction and lateral flow nucleic acid biosensor.

Add 100μL ddH20 to the production of the step above, test the solution by test paper.

AsCas12a and LbCas12a Expression and Purification

AsCas12a and LbCas12a Expression

Plasmid construction

AsCas12a and LbCas12a fragments PCR

LbCas12a fragment:

pET-28a(+) plasmid linearization by PCR.

Electrophoresis the PCR products with 1% agarose gel at 130V for 25 minutes. Check images under UV light and glue recovery.

Then cut them according to the following system and incubate at 37 ℃ for 1 hour.

pET-28a(+) plasmid:

AsCas12a:

LbCas12a:

Next connect AsCas12a and LbCas12a with linearized pET-28a(+) plasmid respectively. Incubated at 37 ℃ for 1 hour.

linearized pET-28a(+) plasmid and AsCas12a fragment:

linearized pET-28a(+) plasmid and LbCas12a fragment:

Use 5μL above reaction system for transforming 50μL E.coli BL21 competent bacteria respectively. Culture at 37℃ for 12-16 hours. Then select 10 single colonies each and put them into the PCR system respectively to PCR.

Electrophoresis the PCR products with 1% agarose gel at 130V for 25 minutes. Check image under UV light. At last, select three strains each and verify them by TSINGKE Biologocal Technology Institute sequencing.

Inducible expression

Culture recombinant BL21 strains to an OD600nm of 0.6-0.8 at 200rpm at 37℃. Then induce protein expression with 0.13mM IPTG and culture them at 120 rpm at 18℃ for 12-16 hours. Next, centrifuge the solution at 6000g for 10 minutes. Abandon supernatant and resuspended precipitation with 10mM imidazole binding buffer. Ultrasonic crushing bacteria, centrifuge the solution at 12000g for 10 minutes and take supernatant for purtification.

Ni-NTA affinity purtification

Pack gravity-flow column with an appropriate amount of Ni-NTA resin. Allow storage buffer to drain from resin by gravity flow. The column volume is 3mL. Balance the column with 6mL 10mM imidazole Wash Buffer. Prepare sample by mixing protein extract with 10mM imidazole Wash buffer so that the total volume is 6mL. Add samples to the column and collect the flow-through. Wash column with 6mL Wash buffer and collect the flow-through. Repeat this step. Elute His-tagged proteins from the resin with 3mL Elution Buffer and collect the flow-through. Repeat this step twice.

Protein analysis

SDS-PAGE

Electrophoresis 16μL above samples and 4μL 5× loading buffer with 12% SDS-PAGE at 70/140V for 60 minutes. Coomassie bright blue stain for 1 hour. Then decolorize overnight.

Western Blot

Transfer proteins to PVDF membrane at 250mA for 2 hours. 5% skim milk seals at room temperature for 1 hour. Then Mouse-anti-His IgG incubates overnight at 4℃. Next, HRP-linked Goat-anti-Mouse IgG incubates at room temperature for 1 hour. Finally, drop in color rendering solution.

BCA kit

Measure absorption values at A_562nm and draw standard curve according to BCA kit protocol. Calculate protein concentrations.

Activity assay

Incubate at 37℃ for 2 hours.

Team:ZJU-China/Experiments

Detect HPV with Menstrual Blood

Pad's blood absorption experiment